Multiplex Assay Kit for 25-Hydroxyvitamin D3 (HVD3) ,etc. by FLIA (Flow Luminescence Immunoassay)

25-HVD3; 25HVD3; Calcifediol; Calcidiol; H-VD3; 25-Hydroxycholecalciferol; 25-Hydroxy Vitamin D3; 25(OH)D

(Note: Up to 8-plex in one testing reaction)

Specificity

This assay has high sensitivity and excellent specificity for detection of 25-Hydroxyvitamin D3 (HVD3) ,etc. by FLIA (Flow Luminescence Immunoassay).
No significant cross-reactivity or interference between 25-Hydroxyvitamin D3 (HVD3) ,etc. by FLIA (Flow Luminescence Immunoassay) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of 25-Hydroxyvitamin D3 (HVD3) ,etc. by FLIA (Flow Luminescence Immunoassay) and the recovery rates were calculated by comparing the measured value to the expected amount of 25-Hydroxyvitamin D3 (HVD3) ,etc. by FLIA (Flow Luminescence Immunoassay) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 95-105 102
EDTA plasma(n=5) 93-101 96
heparin plasma(n=5) 93-101 96
sodium citrate plasma(n=5) 92-103 97

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level 25-Hydroxyvitamin D3 (HVD3) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level 25-Hydroxyvitamin D3 (HVD3) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of 25-Hydroxyvitamin D3 (HVD3) ,etc. by FLIA (Flow Luminescence Immunoassay) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 96-105% 93-101% 83-104% 98-105%
EDTA plasma(n=5) 95-103% 94-102% 97-105% 80-98%
heparin plasma(n=5) 83-93% 94-105% 96-105% 95-104%
sodium citrate plasma(n=5) 88-102% 81-101% 80-89% 99-105%

Stability

The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Reagents and materials provided

Reagents Quantity Reagents Quantity
96-well plate 1 Plate sealer for 96 wells 4
Pre-Mixed Standard 2 Standard Diluent 1×20mL
Pre-Mixed Magnetic beads (22#:HVD3) 1 Analysis buffer 1×20mL
Pre-Mixed Detection Reagent A 1×120μL Assay Diluent A 1×12mL
Detection Reagent B (PE-SA) 1×120μL Assay Diluent B 1×12mL
Sheath Fluid 1×10mL Wash Buffer (30 × concentrate) 1×20mL
Instruction manual 1

Assay procedure summary

1. Preparation of standards, reagents and samples before the experiment;
2. Add 50μL standard or sample to each well,
    add 10μL magnetic beads,and 50μL Detection Reagent A,incubate 60min at 37°C on shaker;
3. Wash plate on magnetic frame for three times;
4. Add 100μL prepared Detection Reagent B, and incubate 30 min at 37°C on shaker;
5. Wash plate on magnetic frame for three times;
6. Add 100μL sheath solution, swirl for 2 minutes, read on the machine.

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Magazine Citations
Allergy & Clinical Immunology Decreased serum LL-37 and vitamin D3 levels in atopic dermatitis: relationship between IL-31 and oncostatin M Wiley: source
Tianjin Med J 成人哮喘血清维生素D 水平与肺功能相关因素分析 Jwk_Tjyy: Cn
Journal of Functional Foods Comparative effects of brown and golden flaxseeds on body composition, inflammation and bone remodelling biomarkers in perimenopausal overweight women S1756464617301561
J Nutr Biochem. Chronic maternal calcium and 25-hydroxyvitamin D deficiency in Wistar rats programs abnormal hepatic gene expression leading to hepatic steatosis in female offspring. pubmed:28219837
 in vivo Effect of Calcitriol on FGF23 Level in Healthy Adults and its Dependence on Phosphate Level pubmed:28064234
Asia pacific allergy Serum 25-hydroxyvitamin D inversely associated with blood eosinophils in patients with persistent allergic rhinitis. pubmed:29094019
Biomed Research International  Effects of Vitamin D Restricted Diet Administered during Perinatal and Postnatal Periods on the Penis of Wistar Rats Pubmed:29850540
Clin Implant Dent Relat Res. Evaluation of FGF‐23 and 25(OH)D3 levels in peri‐implant sulcus fluid in peri‐implant health and diseases Pubmed: 31407857
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Circulating Osteocyte‐Related Biomarkers (vitamin D, sclerostin, dickkopf-1), hepcidin, and oxidative stress markers in early breast cancer: their impact in … Pubmed: 33065276
The Journal of Obstetrics and Gynecology of India The Effects of Follicular Fluid 25 (OH) D Concentration on Intrafollicular Estradiol Level, Oocyte Quality, and Fertilization Rate in Women Who Underwent IVF Program
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